The long-term goal of this project is to investigate the fundamental effects of cell-free hemoglobin solutions (hemoglobin-based oxygen carriers, HBOCs, used as blood substitutes) at the level of the microcirculation, using combined experimental and theoretical approaches. We will examine transport of both oxygen and nitric oxide (NO) in the presence of HBOCs. HBOCs will be used to restore blood and tissue oxygenation under combined reduction of the oxygen supply by isovolemic hemodilution and increased oxygen demand by muscle contraction. The oxygenation of capillaries perfused with red blood cells (RBCs) and those not perfused with RBCs will be measured; we will assess tissue oxygenation through HBOC perfusion of both types of capillaries. Since free hemoglobin is known to be a scavenger of NO, we will investigate transport of nitric oxide in the presence of HBOCs. Nitric oxide concentration and hemoglobin extravasation from microvessels will be measured experimentally. Experiments will be conducted in the hamster retractor muscle and rat spinotrapezius muscle and mesentery; these animals have different hemoglobin oxygen affinities. We will determine the relative contributions of convection and diffusion in increasing tissue oxygenation in the presence of HBOCs and the relative roles of arterioles, capillaries and venules as sources of oxygen. Plasma and tissue PO2 will be measured using phosphorescence quenching microscopy, hemoglobin oxygen saturation in microvessels will be measured microspectrophotometrically, and microvascular geometry and hemodynamic parameters will be measured using video imaging techniques. RBC-free capillaries will be visualized using several optical techniques (phosphorescence probes and fluorescently labeled tracers). We will use a new fluorescence (optical dye) technique to measure the spatial distribution of NO concentration in and around microvessels, and fluorescently labeled HBOC molecules to quantify leakage from the lumen into the tissue. We will use one HBOC that is a 64 kD cross-linked hemoglobin tetramer and one that is a polymerized, high molecular weight hemoglobin. Biophysically detailed computational models will be developed and the experimentally determined vascular and tissue PO2 and NO concentration will be thoroughly compared with the predictions of the models. The research will have a significant impact on our understanding of gas transport with HBOC transfusions under important clinical conditions. [unreadable] [unreadable]